We then determined the anterior-posterior distribution of LHb-to-midbrain functionalconnectivity by recording from dopaminergic and non-dopaminergic neurons followingoptical stimulation of LHb efferents in th-ires-GFP transgenic mice. Fibers originating fromthe LHb were predominantly localized to the posterior VTA and RMTg and the majority oflight-responsive neurons were non-dopamine neurons located in the RMTg and posteriorVTA (Fig. 1b, Supplementary Fig. 1g,h).Since neurotransmission by LHb neurons may encode information related to aversive stimuliprocessing11, we explored whether exposure to an aversive stimulus altered excitatoryneurotransmission at LHb-to-RMTg synapses. We exposed mice expressing ChR2-EYFP inLHb-to-RMTg fibers to either 0 or 19 unpredictable foot shocks in a single 20-min session.One hour later, we performed whole-cell recordings from RMTg neurons in close proximityto LHb-to-RMTg ChR2-EYFP-positive fibers. Voltage clamp recordings from RMTgneurons from foot shock-exposed mice displayed an increase in the frequency of miniatureexcitatory postsynaptic currents (mEPSCs) compared to non-shocked controls (Fig. 1c).Furthermore, LHb-to-RMTg glutamate release probability was significantly enhancedfollowing shock exposure, as indexed by a reduction in the optically-evoked paired pulseratio (Fig. 1d). We observed no differences in mEPSC amplitude or optically-evokedAMPA/NMDA ratios, measurements of postsynaptic glutamate receptor number or function(Fig. 1c, Supplementary Fig. 2). These data suggest that aversive stimuli exposure enhancespresynaptic transmission from LHb inputs to RMTg neurons.To determine whether optogenetic stimulation of LHb-to-RMTg fibers has behavioralconsequences, we optogenetically stimulated this pathway in behaving mice at 60-Hz as thiswas the mean light-evoked firing rate of LHb neurons in brain slices (Supplementary Fig.1b,c and Supplementary Fig. 3). To determine if optogenetic stimulation of LHb-to-RMTgfibers resulted in passive avoidance behavior, we tested mice in a real-time place preferencechamber. When an experimental mouse crossed over into a counter-balanced stimulated-designated, contextually indistinct side of an open field, light stimulation was constantlypulsed until the mouse crossed back into the non-stimulated designated side (Fig. 2a). Miceexpressing EYFP spent equal times on both sides of the chamber, whereas mice expressingChR2-EYFP spent significantly less time on the stimulated side (Fig. 2a, SupplementaryVideo 1) and made significantly more escape attempts (Supplementary Fig. 4a). There wereno differences in total distance traveled or average velocity between ChR2-EYFP and EYFPmice across the entire session (Supplementary Fig. 4b,c). These data suggest that acuteactivation of LHb-to-RMTg fibers promotes location-specific passive avoidance behavior.While activation of the LHb-to-RMTg pathway induced acute avoidance, we nextdetermined if activation of this pathway produced conditioned avoidance using a standardnonbiased conditioned place preference paradigm. 24 hrs after the last conditioning session,where optogenetic stimulation was paired with a distinct context, ChR2-EYFP-expressingmice showed a significant conditioned place aversion for the stimulation-paired chamber,while the EYFP-expressing mice showed no preference or aversion (Fig. 2b). Thisconditioned place aversion was maintained in the ChR2-EYFP-expressing mice 7 daysfollowing the last conditioning session (Fig. 2c), demonstrating that activity in this pathwayalso promotes conditioned avoidance.To determine if mice would perform an operant response to actively avoid activation ofLHb-to-RMTg fibers, ChR2-EYFP or EYFP expressing mice were placed in chamberswhere they could nose-poke to terminate optogenetic stimulation of LHb-to-RMTg fibers(Supplementary Fig. 5a). ChR2-EYFP-expressing mice learned to nose-poke to terminatelaser stimulation over 3 daily training sessions (Supplementary Fig. 6). Following training,ChR2-EYFP-expressing mice made significantly more active nose-pokes to terminate LHb-Stamatakis and StuberPage 2Nat Neurosci. Author manuscript; available in PMC 2013 February 01.$watermark-text$watermark-text$watermark-text

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to-RMTg activation compared to EYFP-expressing mice (Fig. 3a–c), resulting in asignificant increase in the percentange of time the stimulation was off (percent timestimulation was off: ChR2-EYFP: 47.5 ± 7.1 %; EYFP: 2.8 ± 0.9 %; t(10) = 6.28, p <0.0001). These data demonstrate that LHb-to-RMTg activity can negatively reinforcebehavioral responding.Next, we examined whether LHb-to-RMTg activation disrupted positive reinforcment. Wetrained a separate group of mice to nose-poke to earn liquid sucrose rewards. Followingstable responding, nosepokes to earn sucrose in subsequent test sessions where paired with a2s, 60-Hz LHb-to-RMTg stimulation (Supplementary Fig. 5b). ChR2-EYFP-expressingmice receiving stimuliations made significantly fewer nose-pokes compared to EYFP-expressing mice and took significantly longer to retrieve and consume the rewards (Fig.3c,d; Supplementary Fig. 7, Supplementary Video 2). Importantly, there were no significantdifferences between the two groups in the session prior when nosepokes were not pairedwith LHb-to-RMTg stimulation (t(14) = 1.64, p = 0.12), suggesting that stimulation of thispathway time-locked to an operant response served as a punishment.We found that activation of LHb terminals in the RMTg promotes active, passive, andconditioned behavioral avoidance, suggesting that endogenous activity of LHbglutamatergic inputs to the RMTg conveys information related to aversion. The datapresented here suggest that the LHb’s connection with midbrain GABA neurons is crucialfor promoting these behaviors. Consistent with this, direct excitation of VTA GABAneurons disrupts reward-related behaviors10 and stimulation of VTA GABA neurons orinhibition of VTA dopamine neurons promotes aversion12. Importantly, optogeneticstimulation of LHb terminals in the RMTg suppressed positive reinforcement and supportednegative reinforcement, demonstrating this pathway can bidirectionally effect the samebehavioral response (nose-poking) depending on the task. Dopamine signaling in thenucleus accumbens (NAc) promotes positive reinforcement2,3. Thus, motivated behavior tosuppress activation of the LHb-to-RMTg pathway may also depend on dopamine signalingin the NAc. Although encoding negative consequences requires multiple neural circuits,activation of glutamatergic presynaptic inputs to the LHb13,14 or LHb inputs to the midbrainalone produces aversion. Since LHb projections are phylogenetically well-conserved15,neurotransmission in this pathway is likely essential for survival by promoting learning andsubsequent behavior to avoid stimuli associated with negative consequence.Online MethodsExperimental subjects and stereotaxic surgeryWe grouped housed adult (25–30g) male C57BL/6J mice (Jackson Laboratory, Bar Harbor,ME) until surgery. We anesthetized the mice with 150 mg/kg ketamine and 50 mg/kgxylazine and placed the mice in a stereotaxic frame (Kopf Instruments). We bilaterallymicroinjected 0.4 μL of purified and concentrated AAV (~1012 infections units/mL,packaged and titered by the UNC Vector Core Facility) into the LHb (coordinates fromBregma:−1.7 AP, ± 0.48 ML, −3.34 DV). LHb neurons were transduced with virus codingChR2-EYFP or EYFP under the control of the human synapsin (hsyn) promoter. Followingsurgery, we individually housed the mice. For behavioral experiments, we also implantedmice with a unilateral chronic fiber directed above the RMTg (coordinates from Bregma:−3.9 AP, ± 0.3 ML, −4.8 DV). We performed all experiments 6–8 weeks followingsurgeries. We conducted all procedures in accordance with the Guide for the Care and Useof Laboratory Animals, as adopted by NIH, and with approval of the UNC InstitutionalAnimal Care and Use Committees.Stamatakis and StuberPage 3Nat Neurosci. Author manuscript; available in PMC 2013 February 01.$watermark-text$watermark-text$watermark-text

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Histology, immunohistochemistry, and microscopyWe anesthetized mice with pentobarbital and perfused with phosphate-buffered saline (PBS)followed by 4% paraformaldehyde in PBS. We subjected 40 μm brain sections toimmunohistochemical staining for neuronal cell bodies and/or tyrosine hydroxylase (TH: PelFreeze, made in sheep; Neurotrace: Invitrogen, 640 nm excitation/660 nm emission or 435nm excitation/455 nm emission) as previously described10. We mounted sections andcaptured Z-stack and tiled images on a Zeiss LSM Z10 confocal microscope using a 20x or63x objective. For determination of optical fiber placements, we imaged tissue at 10x on anupright fluorescent microscope. We recorded optical stimulation sites as the location intissue where visible optical fiber tracks terminated.Slice preparation for patch-clamp electrophysiologyWe prepared brain slices for patch-clamp electrophysiology as previously described10,16.Briefly, we anesthetized mice with pentobarbital and perfused transcardially with modifiedaCSF. We then rapidly removed the brains and placed them in the same solution used forperfusion, at ~0°C. We cut sagittal midbrain slices containing the RMTg (200μm) orhorizontal midbrain slices containing the VTA and RMTg (200μm) on a vibratome(VT-1200, Leica Microsystems) and placed the slices in a holding chamber and allowed torecover for at least 30 min before recordings.Patch-clamp electrophysiologyWe made whole-cell voltage-clamp recordings of RMTg neurons as previously described16.Briefly, we back filled patch electrodes (3.0–5.0 MOmega;) for current clamp recordings,with a potassium-gluconate internal solution10. For voltage clamp recordings, we backfilledpatch electrodes with a cesium methansulfonic acid internal solution17. For opticalstimulation of EPSCs, we used light pulses from an LED coupled to a 40x microscopeobjective (1 ms pulses of 1–2 mW, 473 nm) to evoke presynaptic glutamate release fromLHb projections to RMTg. For mEPSCs and optically evoked EPSCs, we voltage-clampedRMTg neurons at −70 mV. For AMPAR/NMDAR experiments the holding potential was+40 mV. We added picrotoxin (100 mM) to the external solution to block GABAA receptor-mediated IPSCs for all experiments. For mEPSCs, we added tetrodotoxin (TTX, 500 nM) tothe external solution to suppress action potential driven release. We calculated the AMPA/NMDA ratio and paired pulse ratio as previously described18. We averaged 6 sweepstogether to calculate both the AMPA/NMDA ratio and the paired pulse ratio. We collectedmEPSCs for 5 minutes or until 300 mEPSCs were collected. To determine where, anterior-posterior, midbrain neurons were light responsive, we injected TH-IRES-GFP mice withhsyn-ChR2-EYFP into the LHb. We voltage-clamped (−70mV) GFP-positive (TH+) andGFP-negative (TH−) midbrain neurons and categorized the cells as light-responsive if a lightpulse resulted in an average evoked current across 6 sweeps of > 20 pA.Shock paradigm for patch-clamp electrophysiologyWe placed mice expressing ChR2-EYFP in the LHb-to-RMTg pathway into standard mousebehavioral chambers (Med Associates) equipped with a metal grid floor capable of deliveryfoot shocks for 20 min. Mice received either 19 or 0 unpredictable foot shocks (0.75 mA,500 ms). We presented shocks with a pseudo-random inter-stimulus interval of 30, 60, or 90s. 1 hr following the end of the session, we anesthetized mice for patch-clampelectrophysiology (described above).In vivo optogenetic excitationFor all behavioral experiments, we injected mice with a ChR2-EYFP or EYFP coding virusand also implanted with a chronic unilateral custom made optical fiber targeted to the RMTgStamatakis and StuberPage 4Nat Neurosci. Author manuscript; available in PMC 2013 February 01.$watermark-text$watermark-text$watermark-text

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as described previously19. 3 days prior to the experiment, we connected mice to a ‘dummy’optical patch cable each day for 30–60 min to habituate them to the tethering procedure.Following the tethering procedure, we then ran mice in the behavioral paradigms (seebelow). We used a 10 mW laser with a stimulation frequency of 60 Hz and a 5 ms lightpulse duration for all behavioral experiments.Real time place-preferenceWe placed mice in a custom-made behavioral arena (50 × 50 × 25 cm black plexiglass) for20 min. We assigned one counterbalanced side of the chamber as the stimulation side. Weplaced the mouse in the non-stimulated side at the onset of the experiment and each time themouse crossed to the stimulation side of the chamber, we delivered a 60-Hz constant laserstimulation until the mouse crossed back into the non-stimulation side. We recordedbehavioral data via a CCD camera interfaced with Ethovision software (Noldus InformationTechnologies). We defined an escape attempt as each time a mouse attempted to climb outof the apparatus. We only scored an attempt if no paws were on the ground.Conditioned place preferenceThe CPP apparatus (Med Associates) consisted of a rectangular cage with a left blackchamber (17 cm × 12.5 cm) with a vertical metal bar floor, a center gray chamber (15 cm ×9 cm) with a smooth gray floor, and a right white chamber (17 cm, × 12.5 cm) with a wiremesh floor grid. We monitored mouse location within the chamber using a computerizedphoto-beam system. The CPP test consisted of 4 days. Day 1 consisted of a preconditioningtest that ensured that mice did not have a preference for one particular side20. On days 2 and3, we placed the mice into either the black or white side of the chamber (counterbalancedacross all mice) and we delivered either 0.5-s of 60-Hz stimulation with an interstimulusinterval of 1 s for 20 mins, or no stimulation. Approximately 4 hrs later, we placed the miceinto the other side of the chamber and the mice received the other treatment. 24 hrs after thelast conditioning session, we placed the mice back into the chamber with all three chambersaccessible, to assess preference for the stimulation and non-stimulation paired chamber. Toassess long-term associations between the stimulation and context, we placed the mice backin the chambers 7 days later.Negative and positive reinforcement proceduresBehavioral training and testing occurred in mouse operant chambers interfaced withoptogenetic stimulation equipment as described previously1. For the negative reinforcementprocedure, we placed mice into the chamber and delivered 500 ms of 60-Hz opticalstimulation with an inter-stimulus interval of 1 s. We trained mice on a fixed ratio (FR1)training schedule, in which each nose-poke resulted in one 20-s period where the laser wasshut off, and the LHb-to-RMTg pathway was not optogenetically activated. In addition, atone and houselight cue turned on for the entire 20 seconds and turned off when the laserstimulation returned. For the positive reinforcement procedure, we food restricted a separategroup of mice to 90% of their free-feeding bodyweight. We then trained mice for onesession per day for 1 hr in the operant chambers on a FR1 schedule (in which each nose-poke resulted in 20 uL of a 15% sucrose solution). In addition, a tone and houselight cueturned on for 2 s. Once the mice reached stable behavioral responding (as determined by 3days of over 100 active nose-pokes that did not vary by more than 20% from the first of thethree days), mice received 2s of 60-Hz optical stimulation time locked to the cue followingeach active nose-poke. For both behaviors, we recorded inactive nose-pokes, but these hadno programmed consequences. In addition, we collected and time-stamped the number ofactive and inactive nose-pokes.Stamatakis and StuberPage 5Nat Neurosci. Author manuscript; available in PMC 2013 February 01.$watermark-text$watermark-text$watermark-text

Figure 2. Activation of LHb inputs to the RMTg produces behavioral avoidance(a) Left: Real-time place preference location plots from two representative mice showing theanimal’s position over the course of the 20-min session. Right: ChR2-EYFP-expressingmice spent significantly less time on the stimulated-paired side (t(10) = 7.90, p < 0.0001). n= 6 mice/group for real-time place preference. (b) ChR2-EYFP-expressing mice spentsignificantly less time in the stimulation-paired chamber compared to the non-stimulation-paired chamber 24 hrs after the last stimulation conditioning session (t(7) = 3.54, p = 0.01).EYFP-expressing did not show a preference (t(7) = 0.57, p = 0.58). (c) ChR2-EYFP-expressing mice spent significantly less time in the stimulation paired chamber compared tothe non-stimulation-paired chamber 7 days after the last stimulation session (t(7) = 3.24, p =0.01). EYFP-expressing mice did not show a preference (t(7) = 0.17, p = 0.86). n = 8 mice/group for conditioned place preference.Stamatakis and StuberPage 8Nat Neurosci. Author manuscript; available in PMC 2013 February 01.$watermark-text$watermark-text$watermark-text

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Figure 3. Activation of LHb inputs to the RMTg produces active behavioral avoidance anddisrupts positive reinforcement(a) Example cumulative records of active nose-pokes made by a ChR2-EYFP and an EYFP-expressing mouse to terminate LHb-to-RMTg optical activation. (b) Average number ofactive nose-pokes from one behavioral session in following training (> 4 days; t(10) = 20.52,p < 0.0001). There was no difference in inactive nose-pokes between the two groups (t(10) =0.29, p = 0.78). n = 6 mice per group. (c) Example cumulative records of active nose-pokesmade by a ChR2-EYFP and an EYFP-expressing mouse when optical stimulation waspaired with the nose-poke to receive a sucrose reward. (d) Average number of active andinactive nose-pokes during positive reinforcement (t(14) = 4.01, p < 0.01). There was nodifference in inactive nose-pokes between the two groups (t(14) = 1.22, p = 0.24). n = 8mice per group.Stamatakis and Stuber Page 9Nat Neurosci. Author manuscript; available in PMC 2013 February 01.$watermark-text$watermark-text$watermark-text

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"The LHb is likely to be essential for survival through the promotion of learning and subsequent activities that lead to avoidance of stimuli associated with negative consequences . For instance , optogenetic activation of the LHb promotes active and passive avoidance behavior in mice ( Stamatakis and Stuber , 2012 ) , while bilateral lesion of the LHb reduces escape and avoidance latencies in rats ( Pobbe and Zangrossi , 2010 ) . In addition , under stressful conditions ( i . "

[Show abstract][Hide abstract]ABSTRACT: Abstract Nicotine is one of the most addictive drugs of abuse. Tobacco smoking is a major cause of many health problems, and is the first preventable cause of death worldwide. Several findings show that nicotine exerts significant aversive as well as the well-known rewarding motivational effects. Less certain is the anatomical substrate that mediates or enables nicotine aversion. Here, we show that acute nicotine induces anxiogenic effects in rats at the doses investigated (0.1, 0.5, and 1.0 mg/kg, i.p.), as measured by the hole-board apparatus and manifested in behaviors such as decreased rearing and head-dipping and increased grooming. No changes in locomotor behavior were observed at any of the nicotine doses given. T-pattern analysis of the behavioral outcomes revealed a drastic reduction and disruption of complex behavioral patterns induced by all three nicotine doses, with the maximum effect for 1 mg/kg. Lesion of the lateral habenula (LHb) induced hyperlocomotion and, strikingly, reversed the nicotine-induced anxiety obtained at 1 mg/kg to an anxiolytic-like effect, as shown by T-pattern analysis. We suggest that the LHb is critically involved in emotional behavior states and in nicotine-induced anxiety, most likely through modulation of monoaminergic nuclei.

"However, there are complex interactions between 5HT neurons within the RN that lead to activation of the 5HT projection from the dorsal raphe nucleus to the amygdala, resulting in anxiety (Amat et al., 2001, 2006; Jasinska et al., 2012; Paul and Lowry, 2013). At the same time, the lHb projection to the VTA, through the rostromedial tegmental nucleus, inhibits the mesolimbic DA projection to the NAc, resulting in anhedonia (Hong et al., 2011; Stamatakis and Stuber, 2012; Lamel et al., 2014). These two effects, taken together, provide a neurobiological substrate for the negative information processing bias that characterizes depression (Disner et al., 2011; Willner et al., 2013). "

[Show abstract][Hide abstract]ABSTRACT: The first half of this paper briefly reviews the evidence that (i) stress precipitates depression by damaging the hippocampus, leading to changes in the activity of a distributed neural system involving, inter alia, the amygdala, the ventromedial and dorsolateral prefrontal cortex, the lateral habenula and ascending monoamine pathways, and (ii) antidepressants work by repairing the damaged hippocampus, thus restoring the normal balance of activity within that circuitry. In the second half of the paper we review the evidence that heightened vulnerability to depression, either because of a clinical history of depression or because of the presence of genetic, personality or developmental risk factors, also confers resistance to antidepressant drug treatment. Thus, although antidepressants provide an efficient means of reversing the neurotoxic effects of stress, they are much less effective in conditions where vulnerability to depression is elevated and the role of stress in precipitating depression is correspondingly lower. Consequently, the issue of vulnerability should feature much more prominently in antidepressant research. Most of the current animal models of depression are based on the induction of a depressive-like phenotype by stress, and pay scant attention to vulnerability. As antidepressants are relatively ineffective in vulnerable individuals, this in turn implies a need for the development of different clinical and preclinical methodologies, and a shift of focus away from the current preoccupation with the hippocampus as a target for antidepressant action in vulnerable patients.